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M/P Biomedical Consultants LLC, Mill Valley, CA and * Department of Molecular Microbiology and Immunology, The Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD
2To whom correspondence should be addressed. E-mail: powanda{at}mpbiomed.com.
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ABSTRACT |
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 TOP ABSTRACT LITERATURE CITED |
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KEY WORDS: • infection • clinical studies • animal studies • cytokines • metabolic sequelae • patterned response • dose proportionate • death predicator • bioterrorism • drugs • vaccines • marketing approval • FDA • animal efficacy rule
Others at this symposium have discussed the interactions of repeated infections and nutrition leading to malnutrition and, all too often, to death (1
,2
). We will limit the presentation to the lessons that may be learned from studying acute, generally short-term infections in previously otherwise healthy humans or animals.
Although the concept that infection adversely affects protein metabolism dates from the 19th century, an accurate measure of the extent of this effect awaited the studies such as those of Shaffer and Coleman in 1909 (3
) and of Coleman and DuBois in1915 (4
). Shaffer and Coleman found that they could reduce, and in some cases eliminate, negative nitrogen balance in typhoid patients by providing diets with high caloric value (60–90 cal/kg), primarily derived from carbohydrate, and total nitrogen from 9 to 15 g. In reference to the 70 kg "model person," this represents between 2 and 3 times the calories and the nitrogen required for maintenance of body weight and nitrogen balance in a healthy young individual. Although there were too few patients in their study to allow statistical evaluation, some interesting observations could be made. Increasing nitrogen intake did not result in increased nitrogen excretion, as long as "excess" calories also were administered. However, given a normal caloric intake, increased nitrogen excretion did occur at high nitrogen intake, which indicates that an increased nitrogen intake was beneficial only in the presence of adequate energy to utilize that nitrogen, perhaps suggesting that some synthetic reactions were occurring.
Coleman and Dubois confirmed that an increased net loss of nitrogen occurred during the febrile periods of infectious diseases and that the loss in nitrogen could not be readily compensated for by a higher intake of calories with only a normal daily protein intake. They also found that in typhoid fever, protein, fat and carbohydrate are metabolized the same as in a healthy state, yielding the same amounts of heat. At the height of fever, they found that basal heat production rises 40 to 50% above normal.
Taken together these two studies raise a question as to the true metabolic costs of infection. Is it the apparent energy expended, as measured by calorimetry by Coleman and DuBois, or is it the nutrient levels shown by Shaffer and Coleman needed to "correct" the metabolic defect, that is, negative nitrogen balance, during the illness? If the latter, might this need for excess calories and excess nitrogen in part explain the slow recovery from nitrogen deficit, even in patients with milder infections than typhoid? Might it also account for the difficulty in remedying the debilitating interaction between infection and malnutrition?
In the 1960s, Beisel and his colleagues at the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID) began a systematic study of the metabolic responses to infection. They studied volunteers who were to be exposed to the bacterium, Francisella tularensis, or a rickettsia, Coxiella burnetti (Q fever) or a virus, sandfly fever virus (5
). Volunteers who became acutely ill after exposure to F. tularensis were treated with streptomycin. All these subjects recovered quickly without complications or sequelae. In those exposed to C. burnetti, upon the onset of fever and symptoms, tetracycline therapy was begun. Again, all subjects recovered. Sandfly fever is a self-limiting disease without apparent sequelae in otherwise healthy individuals. This controlled initiation of infection permitted prospective evaluation by metabolic balance techniques during the period of exposure, as well as during overt infection. None of these agents was expected to cause vomiting or diarrhea that would complicate assessing metabolic balance.
The volunteers were begun on a formula diet for 2 wk before exposure. Balance data were collected for 8 d before exposure and for 14–20 d after exposure. Table 1
shows the average nitrogen loss, an estimated tissue loss based on nitrogen loss and the average weight loss recorded for these volunteers. For comparison, values for these parameters in patients with either severe or moderate typhoid fever from the Coleman and Dubois study are shown. Average fever days have been included as a rough index of the severity of the infection. There appears to be a linear response between the amount of nitrogen lost and the persistence of fever. Beisel et al. (5
) observed that the daily excretion of creatinine rose and fell roughly parallel with fever index (product of h x F > 100°F). In individuals exposed to sandfly fever, who suffered only 3 d of fever above 100°F, it took 10 to 11 d to recover from negative nitrogen balance when they were offered only normal amounts of dietary nitrogen and calories.
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) found that dietary restriction accounted for only 35% of the cumulative nitrogen loss that occurred during infection. They also found that the changes in glucocorticoid metabolism that occur during infection appeared inadequate to explain the extent of the catabolic response observed.
Looking at both the early data and those from Beisel et al. (5
), a number of conclusions can be offered. Negative nitrogen balance is not observed until after fever began, but increases and persists for days to weeks after the febrile phase. Negative nitrogen balance appears to correlate with net loss in body weight. Negative nitrogen balance and weight loss are both the product of reduced food intake as well as of infection-induced increased nitrogen excretion. Diets high in both calories and nitrogen can ameliorate the development of negative nitrogen balance. Asymptomatic individuals do not incur negative nitrogen balance.
The catabolic nature of infections has long been known, but more recently there has been evidence of concomitant anabolism. With regard to nitrogen, the translocation of amino acids from muscle to liver not only leads to increased amino acid degradation and increased urinary nitrogen excretion, but it also permits an increased synthesis of plasma proteins, that of the acute-phase proteins.
Similarly, volunteers were exposed to an infectious dose of Salmonella typhosa (typhoid) anticipated to cause illness in 50% of the study group as part of a vaccine evaluation study conducted by Dr. Herbert Dupont of the University of Maryland (6
). Any volunteer with an oral temperature in excess of 100°F was admitted to the research ward. When the temperature was 103°F or higher for 24 h, a course of oral chloramphenicol was begun. All subjects recovered quickly without complications or sequelae. Of 19 exposed subjects, nine developed typhoid fever. The data were normalized to the day of onset of fever in the central panel of Figure 1
, which shows the sequential changes in plasma transferrin, 
-2-macroglobulin, -1-antitrypsin, haptoglobin and -1-acid glycoprotein for subjects who developed typhoid fever and for those who remained asymptomatic. As with negative nitrogen balance, asymptomatic individuals showed no significant changes in their plasma proteins, whereas those who developed disease showed significant changes in all but -2-macroglobulin. A vast array of other acute-phase proteins also increase concomitantly with the onset of illness. Thus, not all nitrogen that is lost from muscle is excreted; in fact, a considerable amount appears to be used in the synthesis of selected plasma proteins.
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) (Fig. 2
). Plasma from infected individuals was sterile-filtered and a portion heat-treated. There clearly was a factor in the plasma from infected individuals that caused plasma zinc and iron depression in recipient animals and increased amino acid transport into their livers. Plasma taken before the infection became evident, or heat-treated plasma from infected individuals, did not cause changes in serum zinc and iron or increase amino acid uptake by the liver.
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For example, radiolabeled analogs of amino acids were used in rats to assess the different categories of amino acid transport (8
). Cycloleucine is an indicator of neutral amino acid transport at the L site and aminoisobutyrate is an indicator of amino acid transport via the A site. In Table 2
it can be clearly seen that during the peak febrile phase, rats infected with either Streptococcus pneumoniae or Salmonella typhimurium exhibited a marked uptake of amino acids by the liver, in some instances as much as fivefold greater than that of control. Muscle tissue had a reduced uptake, suggesting that it lost amino acids. The data are expressed as dpm/µL of intracellular water, given that both liver and muscle gain water during infection.
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To assess whether one could correlate selected changes in metabolism with severity of disease and/or outcome, rats were used to study the effects of respiratory Klebsiella pneumoniae infection (9
). This disease is not uniformly fatal. Tissue counts of viable microorganisms were maximal in lung and blood within 3 d of exposure. Figure 3
shows that selected metabolic sequelae, including lysozyme activity as an indirect index of granulomatous lesions, appeared to parallel the lung and blood microorganism tally. In fact, when the lung titer at 3 d was compared to these metabolic sequelae, the following relationships were observed. The concentration of zinc, the lysozyme activity and the -2-macrofetoprotein contents of plasma all appeared to exhibit a threshold effect wherein a certain concentration of microorganisms needed to be present before there were significant changes in these parameters. -2-Macrofetoprotein is an acute-phase protein specific to rats and akin to C-reactive protein in humans with regard to the pattern of response. The seemingly linear response of plasma seromucoid concentration (an index of overall acute-phase protein synthesis) seems to suggest that this group of proteins may be differently regulated. If lung burden of microorganisms is related to the severity of disease and the outcome, then there should be differences in metabolic sequelae between survivors and those who go on to die.
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demonstrates that when the pattern of metabolic responses in survivors vs. those that died were compared, much greater changes were seen in all parameters, the concentrations of zinc, of seromucoid and of -2-macrofetoprotein, as well as the lysozyme activity in plasma, in those that died.
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) wherein mortality was associated with more extensive metabolic changes in plasma zinc depression, hepatic amino acid uptake as measured by cycloleucine and in seromucoid and -2-macrofetoprotein production (Fig. 5
). Vaccinated rats exhibited a different pattern of responses. When vaccinated rats were challenged with a dose of a strain of tularemia that is uniformly lethal in nonvaccinated rats, there was no evidence of hepatic amino acid uptake or of seromucoid or -2-macrofetoprotein production (Fig. 6
). There were slight changes in serum and liver zinc concentrations, but these were the inverse of what was observed during infection. If a larger dose of the lethal strain was used to challenge the vaccinated rats, the rats survived but rapidly responded with massive changes in zinc and amino acid flux (Fig. 7
). There was some evidence of increased seromucoid and -2-macrofetoprotein production, but at much lower levels than seen in nonvaccinated rats that died from either high doses of the vaccine or lower doses of the lethal strains.
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,8
,10
) or a gram-negative bacterium (8
), a rickettsia (5
,11
,12
) or a virus (5
,13
), or is respiratory (9
,14
) or systemic, the previously healthy host (and all but the most malnourished individuals) respond to infection with a patterned array of metabolic and physiologic alterations. These alterations appear to occur in proportion to the infectious challenge and the likelihood of death. Because of their widespread occurrence and seemingly ingrained status, these alterations may be presumed to be of survival benefit to the host.
Having recognized this pattern, the investigators at USAMRIID, as well as others, such as Ralph Kampschmidt and his group (15
), came to the conclusion that there must be a common mediator or mediators that initiate and regulate these responses. Plasma transfer experiments showed that there was indeed a filterable, heat-labile transferable factor (or factors) in the blood of infected humans and animals. Studies both in vivo and in vitro demonstrated that a similar factor or factors could be elicited from stimulated phagocytic cells. Although it was initially thought that all these host metabolic response activities could be initiated by a moiety we now call interleukin-1, we now know that interleukin-1 is aided and abetted by other cytokines such as interleukin-6 and tumor necrosis factor.
This multiplicity of mediators may explicate the observation that, whereas all the infectious disease models we examined displayed an overall similar pattern of host responses, there were some quantitative metabolic differences in animals between those who survive and those who die, as well as in infected humans.
The presumed survival value of the early metabolic and physiologic changes in infected individuals has been discussed in detail elsewhere (16
,17
). Rather, we would prefer to focus on plasma proteins and their seemingly programmed alteration during infection and on the potential value of the metabolic sequelae in assessing the safety and efficacy of vaccines and drugs to prevent or treat a wide variety of infections.
What might be the value of these increases in acute-phase plasma proteins? One must remember that the increase occurs before the development of any semblance of specific immunity in an infected individual and that they also occur after injury, seemingly in proportion to the extent of illness or injury. Could these plasma protein displays affect the development of immunity in infection, and conversely prevent inappropriate immune recognition after injury? If so, one might expect these plasma proteins to somehow interact with key cells in the immune system. A survey of the literature conducted some years ago indicated that plasma proteins did in fact interact with macrophages, lymphocytes and fibroblasts (18
). That information is summarized in Table 3
.
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A well-known example of how a deficiency in a plasma protein may lead to disease is that of -1-antitrypsin and the development of pulmonary emphysema, as well as chronic liver disease and hepatocellular carcinoma in adults. -1-Antitrypsin deficiency also is the most common genetic cause of liver disease in children (19
). More recently, there is a growing interest in looking at plasma proteins, such as C-reactive protein, as an index of cardiovascular disease (20
) and there is evidence of ceruloplasmin involvement in the development of both cardiovascular disease (21
) and Alzheimer’s disease (22
). The absence of functional ceruloplasmin also can lead to neurodegenerative disease (23
). Polymorphisms of -2-macroglobulin and -1-antichymotrpsin may influence the risk of Alzheimer’s disease (24
).
Considering that all tissues are bathed in blood, is it time to rethink the role of plasma? Is plasma more than a sewer, not merely a conduit for waste products, nutrients and regulatory factors, but also a matrix whose composition changes as a function of environmental factors? Might these changes in the composition of both large and small plasma molecules be designed to alter tissue and organ function? Simply put, are plasma proteins an active part of the body’s coordinated response to age, growth, exercise, infection, inflammation and injury?
Analytical procedures have progressed so as to be able to handle complex mixtures that allow simultaneous determination of multiple analytes, large molecules as well as small. Moreover, gene manipulation now allows us to subtract as well as add components of interest to experimental animals. It may now be possible to determine whether acute-phase proteins materially aid in maintaining health, ameliorating infection and recovering from injury.
Recently, a start-up company [Ciphergen Biosystems Inc., Fremont, CA (www.ciphergen.com)] proposed using plasma protein arrays in preclinical studies as a measure of potential toxicity of drugs under development. This brings us to our second point of focus, the potential value of the metabolic sequelae in assessing the safety and efficacy of vaccines and drugs to prevent or treat a wide variety of infections.
The respiratory Klebsiella pneumoniae and the Francisella tularensis vaccine and challenge studies in rats clearly showed a proportionality between the extent of the metabolic responses and the likelihood of death. Although reducing lethality is the ultimate goal of developing vaccines, ideally one should be able to obtain protection with few or no adverse reactions. Measuring selected metabolic sequelae along with survival can assist in developing a vaccine with a high benefit/risk ratio and in choosing starting doses for clinical studies.
This patterned array can also be used to screen drugs and biologics. A nuclease-resistant form of polyriboinosinic–polycytidylic acid complex [poly(I)–poly(C)] was undergoing study as a potent interferon inducer for the treatment of viral diseases. Preclinical studies indicated that the complex caused dose- and time-dependent decreases in plasma zinc, increases in hepatic amino acid uptake and alterations in some plasma proteins (25
). Although these metabolic changes indicated the presence of an inflammatory state, the fact that -2-macrofetoprotein levels showed minimal change suggested that the drug was not likely to be lethal under the conditions studied. If parallel studies assessing interferon induction and antiviral activity were conducted, one could have used the combined studies to select a formulation or a dose that gave the best response with fewest side effects.
Most recently, the world has been confronted anew with the possibility of biological weapons being used to create terror and incapacitate or kill large numbers of civilians as well as of military personnel. In such instances, protection against, or treatment for, these induced lethal diseases may have to be used in humans without prior clinical trials and solely on the basis of animal data. In fact, the U.S. Food and Drug Administration has amended its new drug and biological product regulations so that certain human drugs and biologics that are intended to reduce or prevent serious or life-threatening conditions may be approved for marketing based on evidence of effectiveness from appropriate animal studies when human efficacy studies are not ethical or feasible (26
). Under this new rule, certain new drug and biological products used to reduce or prevent the toxicity of chemical, biological, radiological or nuclear substances may be approved for use in humans on the basis of evidence of effectiveness derived only from appropriate animal studies and any additional supporting data.
We, therefore, propose that these cytokine-mediated metabolic sequelae, in conjunction with conventional toxicology and survival studies in animals, can aid in the rational selection of safe and effective drug and vaccine candidates intended to reduce or prevent serious or life-threatening conditions.
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTLITERATURE CITED |
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1. Scrimshaw, N. S., Taylor, C. E. & Gordon, J. E. (1968) Interactions of Nutrition and Infection. WHO monograph series no. 57 1968 World Health Organization Geneva, Switzerland.
2. Mata, L. J., Kromal, R. A., Urrutia, J. J. & Garcia, B. (1977) Effect of infection on food intake and the nutitional state: perspectives as viewed from the village. Am. J. Clin. Nutr. 30:1215-1227.
3. Shaffer, P. A. & Coleman, W. (1909) Protein metabolism in typhoid fever. Arch. Intern. Med. 4:538-600.
4. Coleman, W. & DuBois, E. F. (1915) Calorimetric observations on the metabolism of typhoid patients with and without food. Arch. Intern. Med. 15:887-893.
5. Beisel, W. R., Sawyer, W. D., Ryll, E. D. & Crozier, D. (1967) Metabolic effects of intracellular infections in man. Ann. Intern. Med. 67:744-779.
6. Bostian, K. A., Blackburn, B. S., Wannemacher, R. W., Jr, McGann, V. G., Beisel, W. R. & DuPont, H. L. (1976) Sequential changes in the concentration of specific serum proteins during typhoid fever in man. J. Lab. Clin. Med. 87:577-558.[Medline]
7. Wannemacher, R. W., Jr, DuPont, H. L., Pekarek, R. S., Powanda, M. C., Schwartz, A., Hornick, R. B. & Beisel, W. R. (1972) An endogenous mediator of depression of amino acids and trace metals in serum during typhoid fever. J. Infect. Dis. 126:77-86.[Medline]
8. Wannemacher, R. W., Jr, Powanda, M. C. & Dinterman, R. E. (1974) Amino acid flux and protein synthesis after exposure of rats to either Diplococcus pneumoniae or Salmonella typhimurium. Infect. Immun. 10:60-65.
9. Berendt, R. F., Long, G. G., Abeles, F. B., Canonico, P. G., Elwell, M. R. & Powanda, M. C. (1977) Pathogenesis of respiratory Klebsiella pneumoniae infection in rats: bacteriological and histological findings and metabolic alterations. Infect. Immun. 15:586-593.
10. Powanda, M. C., Cockerell, G. L., Moe, J. B., Abeles, F. B., Pekarek, R. S. & Canonico, P. G. (1975) Induced metabolic sequelae of tularemia in the rat: correlation with tissue damage. Am. J. Physiol. 229:479-483.
11. Powanda, M. C., Kenyon, R. H. & Moe, J. B. (1976) Alterations in plasma copper, zinc, amino acids and seromucoid during Rocky Mountain spotted fever in guinea pigs. Proc. Soc. Exp. Biol. Med. 151:804-807.
12. Powanda, M. C., Machotka, S. V. & Kishimoto, R. A. (1978) Metabolic sequelae of respiratory Q fever in the guinea pig. Proc. Soc. Exp. Biol. Med. 158:626-630.
13. Neufeld, H. A., Powanda, M. C., DePaoli, A., Pace, J. A. & Jahrling, P. B. (1978) Host metabolic alterations during Venezuelan equine encephalitis in the rat. J. Lab. Clin. Med. 91:255-226.[Medline]
14. Berendt, R. F., Knutsen, G. L. & Powanda, M. C. (1978) Nonhuman primate model for the study of respiratory Klebsiella pneumoniae infection. Infect. Immun. 22:275-281.
15. Kampschmidt, R. F. (1981) Leukocytic endogenous mediator/endogenous pyrogen. Powanda, M. C. Canonico, P. G. eds. Infection: The Physiologic and Metabolic Responses of the Host 1981:56-57 Elsevier/North-Holland Biomedical Press Amsterdam, The Netherlands. .
16. Powanda, M. C. (1977) Changes in body balances of nitrogen and other key nutrients: description and underlying mechanisms. Am. J. Clin. Nutr. 30:1254-1268.
17. Powanda, M. C. & Beisel, W. R. (1982) Hypothesis: leukocyte endogenous mediator/endogenous pyrogen/lymphocyte-activating factor modulates the development of nonspecific and specific immunity and affects nutritional status. Am. J. Clin. Nutr. 35:762-768.
18. Powanda, M. C. & Moyer, E. D. (1984) Selected aspects of protein metabolism in relation to reticuloendothelial system, lymphocyte and fibroblast function. Reichard, S. M. Filkins, J. P. eds. The Reticuloendothelial System 7A:331-335 Plenum Press New York, NY. .
19. Perlmutter, D. H. (2000) Liver injury in alpha 1-antitrypsin deficiency. Clin. Liver Dis. 4:387-408.[Medline]
20. Ridker, P. M., Rifai, N., Clearfield, M., Downs, J. R., Weis, S. E., Miles, S. & Gotto, A. M., Jr (2001) Measurement of C-reactive protein for targeting of statin therapy in the primary prevention of acute coronary events. N. Engl. J. Med. 344:1959-1965.
21. Fox, P. L., Mazunder, B., Ehrenwald, E. & Mukhopadhyay, C. K. (2000) Ceruloplasmin and cardiovascular disease. Free Radic. Biol. Med. 28:1735-1744.[Medline]
22. Waggoner, D. J., Bartnikas, T. B. & Gitlin, J. D. (1999) The role of copper in neurodegenerative disease. Neurobiol. Dis. 6:221-230.[Medline]
23. Hellman, N. E., Kono, S., Miyajima, H. & Gitlin, J. D. (2002) Biochemical analysis of a missense mutation in aceruloplasminemia. J. Biol. Chem. 277:1375-1380.
24. McGeer, P. L. & McGeer, E. G. (2001) Polymorphisms in inflammatory genes and the risk of Alzheimer’s disease. Arch. Neurol. 58:1790-1792.
25. Powanda, M. C., Sammons, M. L. & Stephen, E. L. (1977) Systemic metabolic alterations associated with repeated injections of a modified polyriboinosinic-polyribocytidylic acid complex. Antimicrob. Agents Chemother. 12:602-605.
26. Anonymous, (2002) New drug and biological drug products: evidence needed to demonstrate effectiveness of new drugs when human efficacy studies are not ethical or feasible. Fed. Regist. 67(31 May):37988-37998.[Medline]
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